The long term goals of this research are characterization of the nature of DNA relaxation by eukaryotic DNA topoisomerase I at a molecular level and identification of those parameters of topoisomerase I inhibition that are critical for the expression of anti-tumor activity. This should permit the identical of second generation topoisomerase I inhibitors suitable for development as anti-tumor agents. For the period of requested support, the specific aims include better definition of the nature of inhibitor binding by the topoisomerase I-DNA covalent binary complex, the identification of an optimal system for studying topoisomerase I-DNA interaction at high resolution and exploration of the possible role of other cellular factors in the expression of cytotoxicity by topoisomerase I inhibitors.